Protein from urine named component B

ABSTRACT

PCT No. PCT/EP93/03645 Sec. 371 Date Jan. 22, 1996 Sec. 102(e) Date Jan. 22, 1996 PCT Filed Dec. 21, 1993 PCT Pub. No. WO94/14959 PCT Pub. Date Jul. 7, 1994A new protein is described obtainable from urine through an extraction and purification process by ion-exchange chromatography and high resolution chromatography.

This is a national stage application of PCT/EP93/03645 filed Dec. 21,1993 and claims benefit to Italian application number RM92A00919 filedDec. 22, 1992.

FIELD OF THE INVENTION

The present invention relates to a new protein named Component B. Inparticular the invention relates to a new protein obtainable from urine,its preparation from urine, its production by recombinant DNA techniquesusing genomic DNA or cDNA encoding said new protein, as well aspharmaceutical compositions containing it and its use in therapy.

SUMMARY OF THE INVENTION

A new protein was isolated during the extraction and purificationprocess of urine derivatives. This protein shows a polypeptide natureand relatively low molecular weight. When human urine is treated withadsorbing materials, as kaolin, and then undergoes filtration, ionexchange chromatography and high resolution chromatography, preferablyaccording to the process hereafter described, after lyophilisation acompound is obtained as amorphous white powder, moving as an a singlepeak in high pressure reversed phase liquid chromatography (HPLC-RP) andhaving a molecular weight of about 9 KDa when analysed byelectrophoresis on a polyacrylamide gel in the presence of sodiumdodecyl sulphate (SDS-Page) under reducing conditions. This protein wasnamed, and is referred to hereinafter as, Component B.

Component B is more specifically characterised through the amino acidsequence reported as SEQ ID NO: 1.

The present invention makes therefore available a new protein, namedComponent B, obtainable through a process comprising the isolation of araw fraction of the compound itself from a dialysed concentrate of urineafter treatment with an adsorbing agent and its purification by ionexchange chromatography and high resolution chromatography as describedhereafter.

Preferably the protein according to the present invention is extractedfrom human urine because of the high amount available. This technique isthus useful for industrial production. The present invention refersparticularly to a polypeptide comprising the SEQ ID NO: 1, its salts,functional derivatives, precursors and active fractions as well as itsactive mutants, i.e. other proteins or polypeptides wherein one or moreamino acids of the structure were eliminated or substituted by otheramino acids or one or more amino acids were added to that sequence inorder to obtain polypeptides or proteins having the same activity ofComponent B and comprises also the corresponding fusion proteins i.e.polypeptides comprising Component B or a mutation thereof fused withanother protein and having a longer lasting half-life in body fluids.Component B can therefore be fused with another protein such as, forexample, an immunoglobulin.

The definition "salts" as used herein refers both to salts of thecarboxyl-groups and to the salts of the amino functions of the compoundobtainable through known methods.

The salts of the carboxyl-groups comprise inorganic salts as, forexample, sodium, potassium, calcium salts and salts with organic basessuch as those formed with an amine as triethanolamine, arginine orlysine. The salts of the amino groups comprise, for example salts withinorganic acids such as hydrochloric acid and with organic acids such asacetic acid. The definition "functional derivatives" as herein usedrefers to derivatives which can be prepared from the functional groupspresent on the lateral chains of the amino acid moieties or on theterminal N- or C-groups according to known methods and are comprised inthe invention when they are pharmacetically acceptable i.e. when they donot destroy the protein activity or do not impart toxicity to thepharmaceutical compositions containing them.

Such derivatives include for example esters or aliphatic amides of thecarboxyl-groups and N-acyl derivatives of free amino groups or O-acylderivatives of free hydroxyl-groups and are formed with acyl-groups asfor example alcanoyl- or aroyl-groups.

The "precursors" are compounds which are converted into the Component Bin the human or animal body. As "active fractions" of the protein thepresent invention refers to any fragment or precursor of thepolypeptidic chain of the compound itself, alone or in combination withrelated molecules or residues bound to it, for example residues ofsugars or phosphates, or aggregates of the polypeptide molecule whensuch fragments or precursors show the same activity of Component B as amedicament.

The present invention refers also to a mixture of polypeptides andderivatives as said above.

A second aspect of the present invention concerns the process ofpreparation or Component B, such process comprising the isolation of araw fraction or the protein from a dialysed concentrate of urine aftertreatment with an adsorbing agent and its purification through ionexchange chromatography and high resolution chromatography.

Preferably, Component B is prepared through the process illustrated inFIG. 1 and comprising the following steps:

a) adsorption of urine at acid pH on kaolin and extraction with ammonia

b) elution of fraction (a) on Bio Rex 70 resin with ammonia

c) elution of fraction (b) on DEAE Sepharose resin with acetate buffer

d) elution of fraction (c) on CM Sepharose ion exchange resin withacetate buffer

e) elution of fraction (d) on HPLC C18 ion exchange resin with a mixtureof acetate buffer and acetonitrile

f) elution of fraction (e) on DE-52 ion exchange resin with acetatebuffer

g) elution of fraction (f) on D-Zephyr ion exchange resin with acetatebuffer

h) elution of fraction (g) on HPLC C18 ion exchange resin with a mixtureof aqueous trifluoroacetic acid and acetonitrile

i) elution of fraction (h) on D- Zephyr ion exchange resin with acetatebuffer.

The present invention refers also to recombinant DNA molecules whichcomprise the nucleotidic sequence encoding the polypeptide according tothe invention, its active mutants or fusion proteins, expression vectorswhich comprise it, host-cells transformed with such vectors and aprocess of preparation of such polypeptide, its active mutants or fusionproteins, through the culture in appropriate culture media of saidtransformed cells. The definition "recombinant DNA molecules" includegenomic DNA, cDNA, synthetic DNA and combinations thereof. In particularthe present invention refers to the nucleotide sequences illustrated inSEQ ID NO: 2 and SEQ ID NO: 3 respectively.

SEQ ID NO: 2: reports the genomic DNA sequence encoding Component B;FIG. 2 reports the restriction map of Component B transcriptional unit;

SEQ ID NO: 3: reports the cDNA sequence encoding Component B; FIG. 8shows the complete Component B cDNA sequence, in which the restrictionsites are indicated. The cloning of Component B can be performed throughdifferent techniques. According to one of these techniques anoligonucleotide, or a mixture of oligonucleotides, are prepared, theirsequence being derived from the sequence of Component B or its fragmentand used as probe for cloning the cDNA or the genomic DNA encodingComponent B.

SEQ ID NO: 4: reports the amino acids sequence encoded both by thegenomic DNA reported in SEQ ID NO: 2 and by the cDNA reported in SEQ IDNO: 3.

The present invention also refers to recombinant DNA molecules whichhybridize with the DNA sequence coding for Component B or fragmentsthereof.

The gene can contain, optionally, the natural introns and can beobtained for example by extraction from appropriate cells andpurification with known methods. Appropriate preparations of DNA, ashuman genomic DNA, are cut in the appropriate way, preferably withrestriction enzymes, find the so obtained fragments are introduced inappropriate recombinant vectors in order to form a DNA library. Suchvectors can be selected with synthetic oligonucleotide probes in orderto identify a sequence encoding Component B according to the invention.

In particular, according to the present invention, the genomic DNA ofComponent B was isolated and cloned.

On the other hand, the corresponding mRNA can be isolated from the cellsexpressing Component B and used to produce the complementary DNA (cDNA)with known methods. This cDNA after having been converted in the doublehelix, can be introduced in an appropriate vector which can afterwardsbe used for transforming an appropriate host cell. The resultingcultures are then selected with an appropriate probe in order to obtainthe cDNA encoding the targeted sequences. Once the wanted clone isisolated, the cDNA can be manipulated essentially in the same way as thegenomic DNA.

The cDNA does not contain introns.

Because of the degeneration of the genetic code, various codons can beused for encoding a specific amino acid, so that one or moreoligonucleotides can be produced, each of them being able to encodefragments of Component B. However only one member of this pool possessesthe nucleotide sequence identical to that of the gene. Its presence inthe pool and its capacity of hybridizing with the DNA also in thepresence of other members of the pool makes it possible the use of thegroup of non fractioned oligonucleotides in the same way as a singleoligonucleotide could be used for cloning the gene encoding the targetedpeptide. Alternatively, a single oligonucleotide containing the sequencewhich is theoretically the most probable for encoding the geneticfragments of Component B (according to what described in the "rules forthe use of codons" in Lathe R., et al. J. Molec. Biol. 183:1-12 (1985))allows the identification the complementary DNA encoding Component B ora fragment thereof.

The processes for hybridizing the nucleic acids are known and described,for example in Maniatis T. et al. Molecular Cloning: A laboratorymanual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1982) and inHaymes B. T. et al. Nucleic Acid Hybridization: A practical approach,IRL Press, Oxford, England. (1985). Through the hybridization using saidprobe or group of nucleotide probes it is possible to identify in agenomic or cDNA gene library the DNA sequences capable of suchhybridization which are thereafter analysed to confirm that they encodethe polypeptide according to the invention (i.e. Component B). Theoligonucleotide which contains such complementary sequence can besynthetized and used as a probe to identify and isolate the gene of thepolypeptide according to the invention i.e. Component B (Maniatis T. etal. ibid.).

Once the appropriate oligonucleotide specific for Component B isselected using the above said method, it is possible to synthetize andhybridize it with a DNA, or preferably with a cDNA derived from cellscapable of expressing the wanted gene preferably after the source ofcDNA was enriched of wanted sequences, for example by extraction of theRNA from cells producing high levels of the wanted gene and conversionof the RNA into the corresponding cDNA using the enzyme reversetranscriptase.

Alternatively, the suitable oligonucleotides specific for Component Bcan be synthesised and used as primers for the amplification ofComponent B cDNA fragments by RACE-PCR (M. A. Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, 1990).

In particular, according to the present invention, a screening ofdifferent human and cellular tissues was performed firstly in order toidentify the best available source for mRNA of Component B. Humantissues from brain, kidney, liver, lung, heart, pancreas, placenta,spleen, testis, thymus and uterus as well as epitheliod carcinoma,promyelocytic leukemia, breast adenocarcinoma, Burkitt's lymphoma andmyeloma cell lines were screened for this purpose.

The screening was performed by using a sensible assay "reversetranscriptase--polymerase chain reaction" (RT-PCR).

Human uterine tissue provided the best source of mRNA.

The cDNA clones of Component B were obtained by said tissue using theamplification method named "3' and 5' rapid Amplification of cDNA Ends"(RACE).

The DNA molecules encoding Component B, obtained with the above saidmethod, were introduced in expression vectors constructed with knowntechniques (Maniatis T. et al. ibid.). The double helix cDNA is ligatedto plasmid vectors using, for example, techniques comprising the use ofsynthetic DNA adapters or techniques of binding "blunt-ended".

For the expression of a targeted protein, an expression vector shouldcomprise also specific nucleotide sequences containing the informationregulating transcription and translation bound to the DNA encoding thedesired protein in such a way that the expression of the gene and theproduction of the protein are permitted. First of all, in order that thegene can be transcribed, it must be preceded by a promoter which can berecognised by the RNA polymerase and to which the polymerase binds, thusstarting the transcription process.

Many promoters are known which operate with different efficency (strongand weak promoters) which are different if used in prokaryotic oreukaryotic cells.

The promoters which can be used in the present invention can beconstitutive, as for example promoter int of lambda bacteriophage,promoter Bla of the gene of β-lactamase of pBR322 and the promoter CATof the gene of chloramphenicol acetyltransferase of pPR325 ecc., orinducible as for example the promoters of prokaryotes as the main rightand left promoters of lambda bacteriophage (P1 and Pr), the promoterstrp, rec A, lac Z, lac I, ompF and gal of E. coli, or the hybridpromoter trp-lac, etc. (Glik B. R. J. Ind. Microbiol. 1:277-282 (1987).

Together with the strong promoters which are capable of producing hugequantities of mRNA, giving high levels of gene expression in prokaryoticcells, it is necessary to use also binding sites for the ribosomes inorder to assure that the mRNA is efficently translated.

An example is given by he Shine-Dalgarno (SD) sequence positioned inappropriate way from the starting codon.

For eukaryotic host cells different sequences regulating thetranscription and translation can be used according to the nature of thehost.

These can be derived from viral sources, as adenovirus, or papillomavirus, Simian virus or similar, wherein the regulation signals areassociated with a specific gene having a high level of expression.Possible examples are the promoter TK of the herpes virus, the promoterof SV40, the promoter of gene gal4 of yeast ecc. The signals regulatingthe starting of transcription can be suitably chosen in order to producerepression or activation in such a way that the expression of the genescan be accordingly modulated.

The DNA molecule comprising the nucleotide sequence encoding Component Bof the invention together with the signals regulating transcription andtranslation are introduced in a vector which is capable of integratingthe sequences of the targeted gene in the host cell chromosome.

The cells which bear the introduced DNA in their chromosome can beselected also introducing one or more markers which make it possible toselect the host cells containing the expression vector. The marker canprovide the cells, for example, with antibiotic resistance or heavymetal (as copper) resistance. The selection gene can be directly boundto the DNA sequences which must be expressed or can be introduced in thecell itself by cotransfection. Other elements may also be necessary fora higher gene expression. These elements can comprise for exampletranscription enhancers and termination signals and introns. Expressionvectors which include such elements comprise those described by OkayamaH. Mol. Cell. Biol. 3:280 (1983).

Among the factors to be considered for chosing a particular plasmid orviral vector are: the facility of detection of the cells containing thevector which can be easily separated from those which do not contain it;the number of copies of vectors which are wanted in a specific host, andthe possibility, or not, of transferring the vector among different hostcells.

The preferred prokaryotic vectors comprise plasmids as those capable ofreplication in E. coli, as pBR322, ColE1, pSC101, pACYC 184 etc.(Maniatis T. et al, ibid.), Bacillus plasmids as pC194, pC221, pT127etc. (Gryczan T. M. The Molecular Biology of the Bacilli, Academicpress, N.Y., 307-329 (1982) Streptomyces plasmids as pIJ101 (Kendall K.J. et al. J. bacteriol. 169:4177-4183) and Pseudomonas plasmids (John J.F. et al. Rev. Infect. Dis. 8: 693-704 (1986) (Izaki K. Jpn. J.Bacteriol. 3: 729-742).

The preferred eukaryotic vectors comprise, for example, BPV, SV40,Baculovirus etc. or their derivatives. Such vectors are known in the art(Bostein D. et al. Miami Wint Symp. 19: 265-274) (Broach J. R. TheMolecular Biology of the Yeast Saccharomyces: Life Cycle andInheritance, Cold Spring Harbor, N.Y., 455-470 (1981) (Broach J. R. cell28:203-204 (1982) (Bollon D. P. et al. J. Clin. Hematol. Oncol. 10:39-48 (1980) (Maniatis T. Cell Biology: A Comprehensive Treatise Vol. 3:Gene Expression Acad. Press N.Y. 563-608 (1980).

The expression vector so prepared is introduced in the appropriate hostcell with an appropriate method such as transformation, transfection,lipofection, conjugation, protoplastic fusion, electrophoration,precipitation with calcium phosphate, direct microinjection etc. Thehost cell which can be used for the present invention can be prokaryoticor eukaryotic cells.

Preferred prokaryotes include bacteria such as E. coli, Bacillus,streptomyces, pseudomonas, Salmonella, Serratia, etc.

Particularly preferred is E. coli, as for example strain 294 of E. coliK12 (AtCC 314446) or E. coli X1776 (ATCC 31537), E. coli W 3110 (F,lambda, ATCC 27325).

Preferred eukariotic host cells are mammalian cells such as human,monkey, mouse or hamster (Chinese Hamster Ovary, CHO) cells since theyassure to the protein molecules post-translation modifications, as forexample the correct folding and glycosylation in the right positions.

Yeast cells can be also used for the present invention. There arevarious recombinant DNA techniques which utilize sequences of strongpromoters and a high number of copies of the plasmid and allow theproduction of the wanted protein in yeast.

After the introduction of the vector in the host cells these arecultivated in a medium which allows the selective growth of cellscontaining the vector.

The expression of the cloned DNA sequence allows the production ofComponent B, or a mutant or fragment thereof. The so expressed proteinis isolated purified through conventional techniques comprisingextraction, precipitation, chromatography, electrophoresis, or similartechniques, or affinity chromatography, using anti-Component Bantibodies immobilised on the column gel. Component B can also beproduced as milk-secreted protein in transgenic animals.

A further aspect of the present invention is the use of Component B, itssalts, functional derivatives, precursors or active fractions as amedicament.

In particular Component B has shown anti-inflammatory, anti-coagulantand anti-tumoral properties. Furthermore Component B can be useful inthe therapy of pathologies correlated with altered levels of TGF-alpha,such as behavioral and hormonal disturbances, angiogenesys, etc.

In fact, Component B has been shown to inhibit the binding of TGF-alphato its receptor with an affinity constant is K_(i) =0.77 * 10⁻¹⁰ Mmeasured by displacement of I¹²⁵ -TGF-alpha from its receptor, obtainedfrom A 431 cell membranes.

The pharmaceutical compositions containing a therapeutically activequantity of Component B in combination with pharmaceutically acceptableexcipients or eluents are also an object of the present invention. Suchcompositions can be formulated for oral, rectal, nasal and particularlyparenteral administration.

Also the topic use of Component B is included in the present invention.

The formulations according to the invention include also forms assubcutaneous implantations based on liposomes or microcapsules ofcopolymers of lactic and glycolic acids.

Other aspects of the invention will be evident in the light of thefollowing detailed description.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Process of preparation of Component B from human urine

The preparation and purification of Component B from human urine issummarized in FIG. 1.

a) STEP 1

The starting material is human urine to which HCl is added up to pH 3.0.After decantation of the precipitate, kaolin is added to the urine (10g/l of starting urine).

The suspension is left for 16 hours and is thereafter centrifuged. Thesupernatant is eliminated and kaolin is extracted with ammonia 2M pH11.0.

The ammonia eluate pH is brought to 8.0 and is concentrated by membraneultrafiltration (cut off 1000 Daltons). The whole operation is performedat 4° C.

b) STEP 2

The solution obtained in step (a) is added with acetic acid up to pH 4.0and then with Bio Rex 70 resin, a weak cation exchange resin, R-COOpreviously equilibrated at pH 4.0 with acetic buffer.

The solution is left under stirring for 4 hours and is then filtered ona pressfilter.

The adsorbed material is eluted from the Bio Rex 70 resin throughelution with ammonia at pH 9.0.

The chromatography eluate is concentrated by membrane ultrafiltration(cut off 1000 Daltons). The whole operation is performed at 4° C.

c) STEP 3

The material obtained in step (b), equilibrated in acetic buffer pH 5,6is adsorbed on an ion exchange resin like DEAE Sepharose, previouslyequilibrated at pH 5,6.

At the end of the adsorption elution is performed using ammonium acetatebuffer 0.5M at pH 5.6. The chromatography eluate is concentrated bymembrane ultracentrifugation (cut off 1000 Daltons). DEAE(diethlaminoethyl) Sepharose is a fast flow weak anion exchange resin.The whole operation is performed at 4° C.

d) STEP 4

The material obtained in step (c) is equilibrated with acetate buffer atpH 4.5 and adsorbed on ion exchange resin like CM (carboxymethyl)Sepharose, a fast flow weak cation exchange resin, previouslyequilibrated at pH 4.5.

When the adsorption is completed elution is performed with ammoniumacetate buffer 0.15M pH 4.5. The chromatography eluate is concentratedby membrane ultrafiltration (cut off 1000 daltons). The whole operationis performed at 4° C.

e) STEP 5

The material obtained in step (d) is purified at 25° C. by reverse phasechromatography on HPLC C18 resin equilibrated in ammonium acetate buffer0.05M pH 5.6.

The adsorbed material is eluted from the resin with an ammonium acetatesolution containing acetonitrile 30% (v/v). The chromatography eluate isconcentrated by distillation (40° C.) under vacuum.

f) STEP 6

The material obtain in step (e) is purified on ion exchange resin likeDE-52, equilibrated at pH 5.6 in ammonium acetate buffer 0.02M.

The elution of the adsorbed material is performed with buffer 0.25M. Theconcentration is performed by membrane ultrafiltration (cut off 1000Daltons). The whole operation is performed at 4° C.

g) STEP 7

The material obtained in step (f) is purified on ion exchange resin likeD-Zephyr, a weak anion exchange resin (substituted tertiary amine)prepacked column (sold by Sepracor), equilibrated at pH 6.2 in 20 mMsodium acetate buffer solution (buffer A).

The elution of the absorbed material is performed by gradient elutionfrom 100% buffer A to 100% 20 mM sodium acetate buffer solution at pH6.2 containing 1M NaCl.

h) STEP 8

The material obtained in step (g) is purified by reversed phasechromatography at 25° C. on resin C18 like HPLC.

After adsorbtion the elution is perfomed with linear gradient formed bya binary mixture of an aqueous solution of trifluoroacetic acid (TFA0.1%) and acetonitrile, acidified with TFA (0.1%). The chromatographyeluate was concentrated by distillation (45° C.) under vacuum andlyophilized.

i) STEP 9

Step 7 is repeated.

The final product, Component B, is recovered as an amorphous whitepowder.

EXAMPLE 2

Analytical characterisation of component B

In order to specify the main physical-chemical characteristics ofComponent B the purified material from urine underwent the followinganalytical controls.

a) AMINO ACID SEQUENCE

The amino acid sequence of Component B was determined according to theEdman method

The analysis was Performed using a sequencer Applied Biosystem, model477A, following the indications given by the producer. Such analysismade it possible to identify the amino acid sequence or component Brelatively to the 81 amino acid residues reported in SEQ ID NO: 1.

b) DETERMINATION OF MOLECULAR WEIGHT

The analysis was performed by "Electron Spray--Mass Spectrometry"(ES-MS) and showed a molecular weight of 8937.9 Daltons. Such analysishas shown the presence of five disulfide bridges and an 80 Daltonsresidue attributable to an SO₄ group bound to Tyr (39).

EXAMPLE 3

Isolation of Human Component B genomic DNA

A human genomic DNA library in lambda phage vector EMBL-3 SP6/T7 waspurchased from Clontech (cat. No. HL 1067 J, Lot No. 1221). Genomic DNAwas extracted from human placenta and partially digested with Sau 3A.DNA fragments were separated on a sucrose gradient to produce a sizerange between 8 to 22 Kb before cloning into the BamHl site of EMBL-3Sp6/T7 vector.

Culture media

E. coli K802 cells, purchased from Clontech (cat. No. C1004-1), werecultured in LB medium supplemented with 10 mM MgSO₄ and 0.2% maltose(culture medium).

The phage library was diluted in 0.1M NaCl, 8 mm MgSO₄, 50 mM Tris-Cl pH7.5, 0.01% gelatin (SM).

The DNA library was plated onto 1.5% agar-LB plates. Top agarose forlibrary plating was: 0.136M NaCl, 0.6% agarose, 1% tryptone (Merck catNo. 7213).

Hybridization reagents

20×SSC 3M NaCl, 0.3M Na citrate, pH 7.0

Hybridization solution 5×SSC, 0.02% SDS, 0.1% N-lauroylsarcosine, 0.5%Blocking reagent (Boehringer cat No. 1096176).

Washing solution A 3×SSC, 0.1% SDS, urea at various concentrationsdepending on the specific probe (HRP-oligos) (see below).

Washing solution B 1×SSC. 0.1% SDS (³² P-oligo CBEX4L)

Detection system

HRP-oligo/DNA hybrids were detected by ECL kit and exposure to HyperfilmECL from Amersham (cat. No. RPM 2106 and 2104, respectively).

³² P-oligoprobed filters were developed by exposure to Hyperfilm β-max(Amersham cat. No. RPN10).

Oligonucleotides

Oligonucleotides were synthesized by an automatic DNA synthesizer(Applied Biosystem mod. 392).

Oligonucleotides were purified by OPC cartridges (Applied Biosystem cat.No. 400771) or by denaturing PAGE.

Oligonucleotides CB1, CB2 and CBEX2L to be used as probes were 5'modified with N-MMT-C12-aminomodifier (Clontech cat. No. 5206-1) duringthe last cycle of synthesis.

Horse-radish peroxidase (HRP, Boehringer cat. No. 814393) was conjugatedto the modified oligonucleotide according to M. S. Urdea (Nuc. Ac. Res.16, 4937, 1988), by using 1,4-phenylendiisothiocyanate (Aldrich cat. No.25,855-5) as a homobifunctional crosslinking agent.

HRP-oligoprobes were purified by anion-exchange HPLC on a NucleopacPA-100 column (Dionex cat. No. 043010). Elution was performed with 20 mMNa phosphate buffer pH 6.0 and a linear gradient of NaCl from 0.2 to1.0M in 30 min..

Purified HRP-oligonucleotides were concentrated by Centricon 10, washedwith PBS and stored at 4° C. in the dark. The HRP-oligonucleotideconcentration was calculated by OD₄₀₃ (ε₄₀₃ =89.5 cm⁻¹ xmM⁻¹).

The following oligonucleotides were synthesized:

    __________________________________________________________________________    oligo #         sequence              target    __________________________________________________________________________    CBPU1         5'TGACTCACACGGCCGGTTCT                               promoter                                    SEQ ID NO: 5    CBPL1         5'CAGCCATGTCCAGTGGTCCT                               promoter                                    SEQ ID NO: 6    CBEX2L         5'ACCACAGCCCATGCTCCA  exon 1                                    SEQ ID NO: 7    CB1  5'TGCAGGAAGCACTGGTCAT exon 2                                    SEQ ID NO: 8    CB2  5'TCTGGCTTGCAGCGGGTAATGGT                               exon 2                                    SEQ ID NO: 9    CB3  5'ATGACCAGTGCTTCCTGC  exon 2                                    SEQ ID NO: 10    CB5  5'ATCCCACCTGCTGCCTTTTG                               intron 2                                    SEQ ID NO: 11    CBEX4U2         5'CGGCGGCTGGGAGCAGT   intron 2                                    SEQ ID NO: 12    CBF1 5'ATGGAATTCTAYCCATTYAAYCARTC                               exon 3                                    SEQ ID NO: 13    CBF2 5'ATGGAATTCTAYCCATTYAAYCARAG                               exon 3                                    SEQ ID NO: 14    CBR1 5'GTAGAATTCGCGCCAATGGARTCNGGRTC                               exon 3                                    SEQ ID NO: 15    CBR2 5'GTAGAATTCGCGCCAATGCTRTCNGGRTC                               exon 3                                    SEQ ID NO: 16    CBEX4U         5'AGTACCCCTTCAACCAGAG exon 3                                    SEQ ID NO: 17    CBEX4L         5'CAGACACCATGAGTGAGCTG                               exon 3                                    SEQ ID NO: 18    CBEX4U1         5'GACACCTCCTCTGTGACGG exon 3                                    SEQ ID NO: 19    CBEX4L1         5'CCAGTTCTGTAGGGTGTCAGT                               exon 3                                    SEQ ID NO: 20    __________________________________________________________________________     where R = AG, Y = CT and N = AG-C-T.

Library titration

The human genomic DNA library was titred according to standardprocedures (F. Ausubel, Current Protocols in Molecular Biology) byinfecting 0.3 ml of an overnight culture of E. coli K802 cells withvarious dilutions of the library, in the range 2×10⁻³ to 2×10⁻⁷.Cell-library mixture was incubated at room temperature for 20 min, thentransferred to 37° C. for 10 min. Infected cells were mixed with 4 ml oftop agarose preheated at 50° C. and poured onto a 10 cm agar plateprewarmed 37° C. Plates were incubated at 37° C. overnight (ON).

The number of plaques was scored in each plate. Duplicate plates wereprepared for each library dilution. The human genomic DNA library titerwas found to be 5×10⁹ pfu/ml, as expected.

Library screning

E. coli K802 cells grown overnight at 37° C. 0.6 ml of cell culture wereinfected with a library aliquot (6×10⁴ pfu) suspended in SM. Infectionand plating was performed as above, but 9 ml of top agarose and 15 cmplates were used.

Semiconfluent plaques were transferred onto Hybond N⁺ nylon membrane(Amersham), according to Amersham instruction manual. flotted DNA wasdenaturated by placing the filters, plaque side up, onto a filter papersoaked in 1.5M NaCl, 0.5M NaOH for 7 min.

Blotted DNA was then neutralized by placing the filters onto a filterpaper soaked in neutralizing solution (1.5M NaCl, 0.5M Tris-Cl pH 7.2, 1mM EDTA) twice for 3 min each.

Filters were washed in 2×SSC and air-dried. DNA was fixed to themembrane by placing the filters onto a filter paper soaked in 0.4M NaOHfor 20 min.

The filters were finally washed in 5×SSC for 1 min and stored in aplastic bag at 4° C. up to hybridization. The human genomic DNA Library(1×10⁶ clones) was screened at high plating density with HRP-CB2oligoprobe. 20 positive clones were selected.

Six positive clones were rescreened with both HRP-CB1 and HRP-CB2oligoprobes. Three clones, named 4D, 12B and 15, were confirmed to bepositive for the gene for Component B.

Hybridization

Filters were preincubated at 42° C. for 30 min in the hybridizationsolution, then hybridized with the appropriate HRP-oligoprobe (5 ng/mloligonucleotide moiety in hybridization solution) at 42° C. for 45 minand finally washed twice for 15 min each at 42° C. in washing solutioncontaining urea at the appropriate concentration (see below).

Filters were washed briefly in 2×SSC at room temperature and thehybridized plaques were detected by ECL reagents and exposure toHyperfilm for 60 min.

Washing conditions for HRP-oligoprobed filters were experimentallydetermined to minimize unspecific hybridization to E. coli and lambdaphage DNAs. Serial dilutions in the range 500 to 15 attomoles of targetDNA were spotted on Hybond N⁺ membrane in the presence of lambda DNA (10ng). Lambda and E. coli DNAs (10 ng each) were used as negativecontrols. Several strips were prepared and used in hybridizationexperiments with 5 ng/ml probe. Washings were performed with washingsolution A containing 0, 9, 18, 27 and 36% urea.

18% and 27% urea were found effective for CB1 and CB2, respectively.Filters hybridized with CBEX2L were washed with washing solution Acontaining 18% urea.

Hybridization with ³² P-oligo CBEX4L was performed at 50° C. and filterswere washed at 45° C. in washing solution B.

Plaque subscreening

Positive plaques were picked up by a Pasteur pipette and transferred toa tube containing 1 ml of SM plus a drop of chloroform. After 2 hrincubation under shaking at room temperature, the phage suspension wasstored at 4° C.

A 10⁻³ dilution of the phage suspension was plated onto a 10 cm plateand rescreened on two replicate filters with two oligoprobes, i.e. theone used in the first screening and another one matching to an adjacentregion of component B.

Independent clones positive for both probes were picked up andresuspended as above.

Preparation of phage stocks

Positive clones were expanded by infecting E. coli K802 cells andgrowing on 15 cm agar plates. After ON incubation at 37° C., theconfluent lysate was collected from agar plates with 10 ml of SM. A fewdrops of chloroform were added, cell debris were removed bycentrifugation at 3000 rpm for 5 min at 4° C. and the clear supernatantcontaining the phages was brought to 50% glycerol, aliquoted and storedat -80° C.

Extraction of phage DNA

2×10⁹ E. coli K802 cells were infected with the selected phage clone(cell/phage ratio=4:1) and grown in 100 ml liquid culture medium ON at37° C. At the end of incubation, full cell lysis was accomplished byadding chloroform (5 μl/ml) to the culture. Phage DNA was extracted byQuiagen, according to the manufacturer's instructions.

Phage DNA sequencing

Phage DNA was sequenced using a cycle sequencing kit from AppliedBiosystem (cat. No. 401388) with an automated DNA sequencer (AppliedBiosystem mod. 373A). Phage DNA was extracted from clones 4D, 12B and 15and sequenced by cycle sequencing.

Sequencing primers were derived either from the amino acid sequence ofComponent B (CBF1, CBF2, CBR1, CBR2) or from the available cDNA orgenomic DNA sequencing data.

Sequencing data indicated that the three clones contained thefull-length Component B gene.

Phage DNA restriction analysis

Phage DNA was subjected to single and multiple restriction enzymedigestions. DNA fragments were resolved by 0.6% agarose gelelectrophoresis and then blotted onto Hybond N⁺ membrane. Filters wererepeatedly probed with oligonucleotides CBEX2L, CB2 and CBEX4L, matchingto exon 1, 2 and 3 respectively.

Subcloning of Component B gene in pBlueScript II SK

Restriction analysis of Component B clone 4D with EcoR1, Xho I and Sfi 1and Southern blotting with oligoprobes specific for the three exons ofComponent B indicated that the entire Component B gene was contained ina 5.2 Kb EcoR1 fragment. (FIG. 5).

Phage DNA was extracted from clone 4D and digested with EcoR1. Theresulting DNA fragments were resolved by agarose gel electrophoresis.The 5.2 Kb fragment was purified by Qiaex (qiagen cat. No. 20020) andligated to EcoR1 linearized pBlueSript II KS (Stratagene cat. No.212207). E. coli strain XL1-Blue (Stratagene cat. No. 200268) wastransformed with the ligation mixture and transformed cells wereselected on Ap/Tc plates. One clone containing the expected plasmid, asshown by resctriction analysis with EcoR1, was isolated and namedpBSCB4D.

Further restriction analysis was performed with Sma1, Kpn1, Hind III,Sfi1, Acc1, Not1, Sal1, Xho1, EcoRV, Cla1, Hinc II, Hind II, Sca1l, BglII, Aat 2, Nco1, Nhe1, Hpa1 and Mlu1. In addition Southern blotting wasperformed with Component B specific oligoprobes on pBSCB4D after singleand double digestions.

FIG. 6 shows the restriction map of pBSCB4D plasmid.

FIG. 4 shows the restriction map of Component B gene. The Component Bgene contains 3 exons separated by 2 introns. The exons are flanked byappropriate consensus acceptor and donor splice sites.

Exon 1 is 84 bp in length and contains 26 nt of untraslated mRNA and thesequence coding for 19 amino acids of a putative signal peptide. It isseparated from exon 2 by an intron of 410 bp.

Exon 2 is 120 bp in length and codes for 3 amino acids of a putativesignal peptide and 37 amino acids of the mature protein. It is separatedfrom exon 3 by an intron of about 550 bp.

Exon 3 is 326 bp long; it encodes the C-terminal 44 amino acids ofComponent B and 197 nt of untraslated mRNA, containing a polyadenylationsignal (TATAAA) 14 bp upstream to the 3' processing site, to which endthe poly(A) tail is attached.

In particular, in the three genomic clones the signal peptide encodingsequence was found to contain a Leu codon at position 11 of the putativesignal peptide.

The amino acid sequence of Component B derived from the genomic gene wasfound to be identical to the one experimentally determined by Edmandegradation.

Sequence analysis upstream to exon 1 evidentiated a promoter region(FIG. 3) and corresponding to nucleotides 1-577 of SEQ ID NO: 2)containing a TATA box at -28 and various upstream promoter elements andenhancers, including a GC-rich box at -58, an AP-1 site at -83, an AP-2site at -360 and several E boxes.

The TATA box is the preferred binding site for the transcriptioninitiation factor TFIID. The GC-rich box represents the binding site forSp-1, a general transcription factor involved in the transcription of awide variety of genes (Transcription and Splicing B. D. Hames & D. M.Glover Eds, IRL press, 1988).

The AP-1 site is the binding site for AP-1, the transcription factorcomplex formed by c-fos and c-jun. The AP-1 site is present in severalgenes involved in cell growth and differentiation. AP-1 is one ofseveral cis-elements that mediate induction responses to activators ofprotein kinase C (The hormonal control of gene transcription P. Cohen &J. G. Foulkes Eds. Elsevier, 1991).

The AP-2 site is the target for AP-2, a transcription factor activatedby PMA and cAMP (ibidam).

E boxes are common sequences found in several enhancer regions and playan important role in determing the tissue-specific expression of genes.E box contains the sequence CANNTG, with the two internal bases changingaccording to the specific E box (R. E. Kingston Current Opinion Cell.Biol. 1989; 1, 1081-1087).

The Component B promoter contains a potential responsive element forglucocorticoid receptor (GRE), which indicates that the Component B genecould be induced by glucocorticoids.

Subcloning of Component B gene in a vector for expression in mammaliancells

It is known that the expression of rec-proteins in mammalian cells maybe improved by the presence of intron(s). The Component B genomic DNAcan be expressed in mammalian cells.

To this end, a 1364 bp fragment spanning Component B gene from +50 to+1413 is excised from pBSCB4D by Pvu II and Nar 1 digestion.

FIG. 2 shows the restriction map of Component B transcriptional unitwhere Pvu II and Nar1 sites are based. The entire Component B gene isreconstituted by ligation of this fragment with a syntheticoligonucleotide reproducing the 5' end of the gene, flanked by asuitable restriction site for the subsequent gene cloning in aneukaryotic expression plasmid.

Example 4: Isolation of Component B cDNA clones

Rapid Amplification of cDNA Ends (RACE), a technique described byFrohman et al., (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8998, was usedto obtain partial cDNA clones corresponding to the 5' and 3' ends of theComponent B mRNA. The partial clones contained overlapping DNA sequencesand thus could be combined to construct the full-length Component B cDNAsequence. A diagram depicting the general strategy used for RACE cloningis shown in FIG. 7.

For 3' RACE, the DNA sequence of the second exon of the Component B genewas available and was used to design the gene specific primer CKCB1(5'-TCAAGTGCTACACCTGCAAGGAG-3') (SEQ ID NO: 21). cDNA synthesis wasprimed from the poly A tail of human uterus poly A⁺ RNA with theoligonucleotide 5'-GGCCACGCGTCGACTAGTAC-3' (SEQ ID NO: 24), called theadapter primer of AP. The cDNA was used as the template for a polymerasechain reaction (PCR) with the CKCB1 and AP primers, which produced anapproximately 450 base pair (bp) fragment corresponding to the 3' end ofthe Component B cDNA.

For 5' RACE, the primer CKCB7 (5'-CGTCAGAGAGGAGGTG-3') (SEQ ID NO: 22was designed from the DNA sequence of the 450 bp 3' RACE fragment andwas used to prime cDNA synthesis from human uterus poly A⁺ RNA.

After purification to remove the mRNA and the CKCB7 primer, anoligodeoxycytidine tail was added to the 3' end of the cDNA. The tailedcDNA was used as the template in a PCR with the nested primer CKCB2(5'-ACCGTCACCAGCGTGGTC-3') (SEQ ID NO: 23) and the anchor primer (ACP,5'-CTACTACTACTAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3') (SEQ ID NO: 25)or a mixture of the ACP and the universal amplification primer (UAP,5'CTACTACTACTAGGCCACGCGTCGACTAGTAC-3') (SEQ ID NO: 26). CKCB2 annealedto the 3' end of the second exon sequence and the ACP annealed to theoligodeoxycytidine tail. An approximately 230 bp fragment was obtainedwhich contained DNA sequence corresponding to the 5' end of theComponent B mRNA.

General experimental protocols (such as polyacrylamide gelelectrophoresis, ethanol precipitation, ligation, and restrictionendonuclease digestion), bacterial culture media (such as LB) andchemical solutions (such as phenol) used are described in detail inSambrook et al, (1989) Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press. New York, unless otherwisereferenced.

3' RACE cloning procedure

The 3' RACE System for Rapid Amplification of cDNA ends was purchasedfrom Life Technologies, Inc., Grand Island, N.Y. Human uterus poly A⁺RNA was purchased from Clontech Laboratories, Inc. Palo Alto, Calif.First strand cDNA synthesis was accomplished using the protocol andreagents supplied with the 3' RACE system. Briefly, 1 μl (1 μg) ofuterus poly A⁺ RNA was combined with 1 μl of a 10 μM solution of AP and12 μl of diethyl pyrocarbonate (DEPC)-treated water and the mixture washeated to 65° C. for 10 minutes. After chilling the mixture on ice, thereaction components were added so that the final composition wasapproximately 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl₂, 100μg/ml bovine serum albumin, 500 nM AP, 500 μM each of dATP, dCTP, dGTP,and dTTP, and 50 ng/μl RNA in a volume of 19 μl. The reaction mixturewas heated to 42° C. and 1 μl (20 units) of SuperScript reversetranscriptase was added. After incubation for 30 minutes at 42° C., thereaction mixture was chilled on ice and 1 μl RNaseH 12 units) was added.RNaseH digestion was conducted for 10 minutes at a temperature of 42° C.The reaction mixture was stored at -20° C. prior to the PCR.

For PCR, four identical 40 μl mixtures were prepared each with thefollowing composition: 1 μl of uterus poly A⁺ cDNA in 40 mM KCl, 70 mMTris-HCl (pH 8.8), 0.1% Triton X-100, 1 mM MgCl₂, 0.25 μM CKCB1, and 0.5μM AP. Reagents from the 3' RACE system were not used for PCR. Both theCKCB1 and AP primers were synthesized on an Applied Biosystems, Inc.model 392 oligonucleotide synthesizer. After deprotection,lyophilization, and resuspension in DEPC-treated water the opticaldensity of each solution was measured at a wavelength of 260 nm. Basedon the optical density measurements, a 10 μM solution of eacholigonucleotide was prepared in DEPC-treated water. The crudeoligonucleotide solutions were used for PCR with no furtherpurification. The concentration of crude AP that produced identicalresults to the AP supplied with the 3' RACE system was experimentallydetermined; 0.4 μM crude AP was equivalent to 0.2 μM AP from LifeTechnologies, Inc. in the PCR.

The 40 μl PCR reactions were heated to 94° C. in a temperature cyclerbefore adding to each a 10 μl mixture containing the following: 1.25units AmpliTaq DNA polymerase (Perkin Elmer Cetus, Norwalk, Conn.), 40mM KCL, 70 mM Tris-HCl, pH 8.8), 0.1% Triton-X-100, 1 mM MgCl₂, and 1 mMeach of dATP, dTTP, dGTP, and dCTP. The final concentration of eachreagent in the PCR was approximately 1 μl uterus cDNA per 50 μl, 1.25units AmpliTaq DNA polymerase per 50 μl, 40 mM KCl, 70 mM Tris-HCl (pH8.8). 0.1% Triton X-100, 1 mM MgCl₂, 0.2 μM CKCB1, 0.4 μM AP, and 0.2 mMeach of dATP, dTTP, dGTP, and dCTP. After completing a 5 minuteincubation at 94° C., a "Touchdown" PCR temperature cycling program wasperformed according to Don, R. H., Cox P. T., Wainwright. B. J., Baker,K. and Mattick, J. S. (1991) Nucl. Acids Res. 19, 4008, by varying theannealing temperature from 73° C. to 63° C.

After PCR amplification, the four reactions were combined and the DNAproducts were size-fractionated by electrophoresis on a 5%polyacrylamide gel. A DNA product of aproximately 450 bp was excisedfrom the gel and purified by electroelution in dialysis tubing. Theeluate was extracted with a 50:50 (v:v) mixture of phenol andchloroform, ethanol precipitated, dried, and resuspended in 10 μlsterile water.

Due to the template independent terminal transferase activity of Taq DNApolymerase and its strong preference for dATP (Clark, J. M. (1988) Nucl.Acids Res. 16, 9677 and Mole. S. E., Iggo, R. D. and Lane D. P. (1989)Nucl. Acids Res. 17, 3319), the purified 450 bp PCR fragment wasexpected to have a single deoxyadenosine residue at each 3' end. Forsubcloning and characterizing the PCR fragment, a pBluescriptSK+(Stratagene, La Jolla. Calif) "T-vector" was prepared essentially asdescribed by Marchuk et al. (1991) Nucl. Acids Res. 19, 1154. ThepBluescript plasmid (20 μg) was digested with EcoRV restrictionendonuclease and then purified by extraction with a 50:50 (v:v) mixtureof phenol and chloroform. After ethanol precipitation, the DNA wastreated with 9 units of Taq DNA polymerase for 2 hours at 70° C. in a 50μl reaction containing 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% TritonX-100, 1.5 mM MgCl₂, and 2 mM dTTP. The vector was again purified byextraction with phenol and chloroform (50:50 v:v) and ethanolprecipitation. This procedure resulted in the addition of a singledeoxythymidine residue to each 3 ' terminus, and rendered the vectorcompatible for insertion of DNA fragments synthetized with Taq DNApolymerase.

The unphosphorylated 450 bp PCR fragment was inserted into the T-vectorin a reaction with T4 DNA ligase (New England Biolabs, Beverly, Mass.)using the conditions recommended by the manufacturer. The ligationreaction was incubated for approximately 72 hours at 16° C. and thenused to transform competent E. coli XL1-Blue cells (Stratagene, LaJolla, Calif.). The transformed cells were plated onto LB agarcontaining 50 μg/ml ampicillin. Prior to plating the cells, 100 μl 2%X-gal (Life Technologies, Inc., Grand Island, N.Y.) and 40 μl 100 mMIPTG (Life Technologies, Inc., Grand Island, N.Y.) were sequentiallyspread onto the agar surface of each plate and allowed to dry. After anovernight incubation at 37° C., colonies with blue pigment andunpigmented colonies (white) were visible. Plasmid DNA was purified fromcultures of 12 white colonies. All 12 isolates contained the 450 bpinsert. Five clones were chosen for further analysis: 3CB4, 3CB6, 3CB7,3CB8 and 3CB9. DNA sequence analysis was done using a Sequenase version2.0 kit (United States Biochemical, Cleveland, Ohio).

5' RACE cloning procedure

The 5' RACE system for Rapid Amplification of cDNA ends was purchasedfrom Life Technologies, Inc.. Grand Island, N.Y. Human uterus poly A⁺RNA was purchased from Clontech Laboratories, Palo Alto, Calif. The 5'RACE cloning experiments were done using the protocol and reagentssupplied with the 5' RACE system with the following exceptions: (a) theCKCB7, ACP and UAP primers were synthetized on an Applied Biosystem,Inc. model 392 oligonucleotide synthetizer and prepared as described for3' RACE cloning, and (b) the "Touchdown" PCR temperature cycling programdescribed for 3' RACE cloning was used for cDNA amplification. Firststrand cDNA was synthetized as follows: 1 μl (1 μg) of uterus poly A⁺RNA was comnbined with 0.5 μl of a 10 μM solution of CKCB7 and 13.5 μlof DEPC-treated water and the mixture was heated at 70° C. for 10minutes. After chilling the mixture on ice, the reaction components wereadded so that the final composition was approximately 20 nM Tris-HCl (pH8.4), 50 mM KCl, 3 mM MgCl₂, 10 mM DTT, 200 mM CKCB7, 400 μM each ofdATP, dCTP, dGTP, and dTTP, and 40 ng/μl RNA in a volume of 24 μl. Thereaction mixture was heated to 42° C. and 1 μl (220 units) ofSuperscript II reverse transcriptase was added. After incubation for 30minutes at 42° C. and for 15 minutes at 70° C., the reaction mixture wasplaced at 55° C. and 1 μl RNaseH (2 units) was added. RNaseH digestionwas conducted for 10 minutes at a temperature of 55° C.

The cDNA was separated from unincorporated dNTPs, CKCB7, and proteinsunsing a Glassmax DNA Isolation Spin Cartridge (included in the 5' RACEsystem). Specifically, 120 μl of binding solution (6M NaI) was added tothe first strard reaction, and the CDNA/NaI solution was transferred toa GLASSMAX spin cartridge. Following centrifugation at 13,000× g for 20seconds, 0.4 ml of cold (4° C.) 1× wash buffer was added. The spincartridge was centrifuged at 13,000× g for another 20 seconds. This stepwas repeated two additional times. After washing twice with 400 μl ofcold (4° C.) 70% ethanol, the cDNA was eluted by adding 50 μl ofsterilized, distilled water to the spin cartridge and centrifuging at13000× g for 20 seconds. A homopolymer tail was added to the 3' end ofthe cDNA using terminal deoxynucleotidyl transferase (TdT) and dCTP. Thetailing reaction was performed in a PCR compatible buffer. 10 μl ofpurified cDNA was combined with 7.5 μl of DEPC-treated water, 2.5 μl of10× reaction buffer, 1.5 μl of a 25 mM solution of MgCl 2 and 2.5 μl ofa 2 mM solution of dCTP. The reaction mixture was incubated for 2 to 3minutes at 94° C. After chilling for 1 minute on ice, 1 μl of TdT (10units/μl) was added. The final composition was therefore: 10 μl cDNA in20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 200 μM dCTP, 0.4units/μl TdT. The reaction mixture was incubated for 10 minutes at 37°C. and then for 10 minutes at 70° C. to inactivate the TdT.

For the PCR, four different reactions were prepared with the 15following final primer concentrations (per 50 μl):

1. 400 nM ACP

2. 800 nM ACP

3. 360 nM UAP and 40 nM ACP (UAP:ACP, 9:1)

4. 720 nM UAP and 80 nM ACP (UAP:ACP, 9:1)

The final concentrations (per 50 μl) of the remaining components in allfour reactions were identical: 5 μl of uterus poly A⁺ dC-tailed cDNA in20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl₂, 400 nM CKCB2, and 200μM each dATP, dCTP, dGTP and dTTP. The components, including the ACP andUAP primers, were mixed in an initial volume of 45 μl and heated to 94°C. in a temperature cycler. A 5 μl mixture of 1.25 units of AmpliTaq DNApolymerase (Perkin Elmer Cetus, Norwalk, Conn.) in 20 mM Tris-HCl (pH8.4) was then added to each reaction to bring the final volume to 50 μl.The "Touchdown" PCR temperature cycling program (as described for 3'RACE cloning) was used to amplify 5' cDNA fragment.

After PCR amplification, the four reactions were combined and the DNAproducts were size-fractionated by electrophoresis on an 8%polyacrylamide gel. A DNA product of approximately 230 bp was excisedfrom the gel, purified, and subcloned into the "T-vector" as describedfor the 450 bp 31 RACE fragment. Five clones were chosen for furtheranalysis: 5CB2, 5CB3, 5CB5, 5CB6, 5CB11. DNA sequence analysis was doneusing a Sequenase version 2.0 kit (United States Biochemical. Cleveland,Ohio).

FIG. 8 reports the complete Component B cDNA sequence assembled fromRACE clones 5CB3 and 3CB7, in which the restriction sites are indicated.

By sequence alignment the cDNA sequence of clones 5cb3, 5cb6, 5cb11 and3cb4, 3cb7, 3cb9 were shown to perfectly match with the Component Bexons (genomic clone 4D of Example 3). Although the present inventionwas illustrated with specific examples, it is clear that modificationscan be performed on the operations as described always remaining withinthe spirit and scope of the invention.

LEGENDS TO FIGURES

FIG. 1. Flow chart of the process of purification of Component B fromurine.

FIG. 2. Restriction map of Component B genomic transcriptional unit(such Component B genomic DNA reported in SEQ ID NO: 2 with the aminoacid sequence of the encoded protein present as SEQ ID NO: 4). Arrowsindicate the splicing sites.

FIG. 3. Sequence of Component B promoter region (said Component Bpromoter region reported in SEQ ID NO: 2). Binding sites for AP-1, AP-2,Sp-1 and E-boxes transcription factors are indicated. TATA box isindicated. GRE is also indicated.

FIG. 4. Restriction map of Component B gene. The derived mRNA is shownbelow the genomic gene by a line, the boxed region represents theprotein encoding sequence.

FIG. 5. Restriction map of clone 4D insert.

FIG. 6. Restriction map of pBSCB4D plasmid.

FIG. 7. General strategy used for RACE cloning of Component B DNAsequence.

FIG. 8. Complete Component B cDNA sequence, in which the restrictionsites are indicated (said Component B cDNA reported in SEQ ID NO: 3).

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 26    - (2) INFORMATION FOR SEQ ID NO: 1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 81 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -     (vi) ORIGINAL SOURCE:              (A) ORGANISM: URINE    #1:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    -      Leu Lys Cys Tyr Thr Cys Lys Glu - # Pro Met Thr Ser Ala Ser Cys    Arg    #   15    -      Thr Ile Thr Arg Cys Lys Pro Glu - # Asp Thr Ala Cys Met Thr Thr    Leu    #                 30    -      Val Thr Val Glu Ala Glu Tyr Pro - # Phe Asn Gln Ser Pro Val Val    Thr    #             45    -      Arg Ser Cys Ser Ser Ser Cys Val - # Ala Thr Asp Pro Asp Ser Ile    Gly    #         60    -      Ala Ala His Leu Ile Phe Cys Cys - # Phe Arg Asp Leu Cys Asn Ser    Glu    #     80    -      Leu    - (2) INFORMATION FOR SEQ ID NO: 2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 2031 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA (genomic)    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: join(578..63 - #5, 1041..1160, 1687..1817)    -     (ix) FEATURE:              (A) NAME/KEY: exon              (B) LOCATION: join(552..63 - #5, 1041..1160, 1687..2012)    -     (ix) FEATURE:              (A) NAME/KEY: mat.sub.-- - #peptide              (B) LOCATION: join(1049..1 - #159, 1686..1817)    -     (ix) FEATURE:              (A) NAME/KEY: TATA.sub.-- - #signal              (B) LOCATION: join(524..52 - #9, 1992..1998)    -     (ix) FEATURE:              (A) NAME/KEY: GC.sub.-- - #signal              (B) LOCATION: 493..498    #/note= "GC-rich box represents the                   binding s - #ite for the transcription factor Sp-1"    -     (ix) FEATURE:              (A) NAME/KEY: promoter              (B) LOCATION: 1..551    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: join(17..22, - # 24..29, 55..60, 66..71, 434..439)    #/standard.sub.-- name= "E-box":    #"E-box site is described in R.E. Kingston                   Current O - #pinion Cell. Biol., 1989, Vol.1,    #(Citation 1) "1081-1087    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #signal              (B) LOCATION: 329..342    #/function= "GlucocorticoidTION:    #element"      responsive                   /standard.sub.-- - #name= "GRE"    -     (ix) FEATURE:              (A) NAME/KEY: protein.sub.-- - #bind              (B) LOCATION: 469..475    #/standard.sub.-- name= "AP-1 site"    #"AP-1 site is the binding site for the                   transcriptio - #n factor AP-1"    -     (ix) FEATURE:              (A) NAME/KEY: protein.sub.-- - #bind              (B) LOCATION: 191..197    #/standard.sub.-- name= "AP-2 site"    #"AP-2 site is the binding site for the                   transcriptio - #n factor AP-2"    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #signal              (B) LOCATION: join(578..63 - #4, 1040..1049)    #/function= "Putative signalION:                   peptide"    -     (ix) FEATURE:              (A) NAME/KEY: intron              (B) LOCATION: join(636..10 - #40, 1161..1686, 2013..2031)    #2:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - TGGCCCATGC TACCCTCACC TGACACCTGC TTCCTACCTC TGGTTTCTAC TT - #TGCAGGTG      60    - TGTATCAGGT GTACACAGAC CAGGTAGAGG TCTGTGGAGA GGGCTGCAGG CC - #AGGCTGCA     120    - GGGAAGGGGT GCCAGGCGGG GCTAGAGCAA CAAGGGCAGA GGCTACACTG AA - #CCTGGGTC     180    - YTAAGGGTCC CCCAGGCTGG GGCTGGGTGG CCTATGTGAA CCCCAGAGGC AC - #AGCCAGGA     240    - CATGGGGGCT CATCAGAGGG GCAGTCTGAG CTCAGCAGGA AAGGCCTTCT CT - #GTCAGAGC     300    - TGTCCCAGGA CCACTGGACA TGGCTGAGGA ACAGTGAGTT CCCCAGTGTT GG - #AGGTGTGC     360    - AAGCAGAGGC CTGGCCATCG TCCTCAGACA CAGCTCCCAG ATCCAGCTCC CT - #GCCCGTCT     420    - GCCATGTTCC TGCCAGCTGC CTCCCCACTG GGCCCTTTAC CACGTTCCTG AC - #TCACACGG     480    - CCGGTTCTGC CACCGCCCAG AAGCCGGTGC CCAAGGGCCT GGCTATAAAT CC - #TTGATGTG     540    #CGC TGG GCT     595TCA CTTCTGAGCA CGGAGCA ATG GCC TCT    #     Met Ala Ser Arg Trp Ala    20    - GTG CAG CTG CTG CTC GTG GCA GCC TGG AGC AT - #G GGC TGT  G GTGAGTGGGC     645    Val Gln Leu Leu Leu Val Ala Ala Trp Ser Me - #t Gly Cys    5    - CGCAGGCTGG TGGGGACCTT GCCTCTGAGC TTGTCTGCCC ACCTCCTAGG GG - #GATGGGGC     705    - TGTTGGGGGT GCTTTGTGGC TGAGAGCCTC CTTAGGCCTC CATGAGGCTC AC - #CCTCCTCA     765    - TTCTCAGTGA GCCTCCTGGG TCCCAGAGCC CAGCTTCACC CTGGGACAGG GG - #TCACGGCT     825    - CCACTCTGCA GGAAGGGAGA CTGAGGCTTG GTGGAGGGAT GCAGCATTCA AG - #TCTGTGGC     885    - TCAGCTCAGT TAGAGAAAGC TGCCAGAGAG GCCCCTTGAA GGSCTGCCCG GG - #GCCTTGAA     945    - AGATGTCAGC GAGACTCCTT CAGCCCCTGC CTCCTGGTTC CAGGATGAGV CC - #ACCGAGGT    1005    #CTC AAG TGC      1057C CCATCCCTCA CCCAG  GT GAG GCC    #   Gly Glu Ala Leu Lys Cys    2       1    - TAC ACC TGC AAG GAG CCC ATG ACC AGT GCT TC - #C TGC AGG ACC ATT ACC    1105    Tyr Thr Cys Lys Glu Pro Met Thr Ser Ala Se - #r Cys Arg Thr Ile Thr    #      15    - CGC TGC AAG CCA GAG GAC ACA GCC TGC ATG AC - #C ACG CTG GTG ACG GTG    1153    Arg Cys Lys Pro Glu Asp Thr Ala Cys Met Th - #r Thr Leu Val Thr Val    # 35    - GAG GCA  G GTGAGGCCAG GCCCCACGGC AGCCCTGGGT GCAGTGG - #AGT CAGGGCCACC    1210    Glu Ala    - TCCCCCAAGT GCGTCCCTCC TTTGCTGGTG CTCCTCCCGG CCCAAAAGGA AG - #CAGGTGGG    1270    - ATGGGCAGAA CAGGCTGCCA CACCTTGGCA GGGGTGCCTT CCACGAGGGT GG - #CACAGCCC    1330    - CCTCAGAGAC CCAGTCCTGG GGCACCAGGC GCTGGAGGTG GGTGGGGCTT AA - #TGGCCGGG    1390    - GTACCCTGGG GGGCTCAAAC CCCAGCTCTG ACACAGACCC ACTGGGTGGT GT - #TGCCACAG    1450    - CCTCTGGGCT CGGGCTCCCA TCTCAGCGCA GGCACTTCAG AGGTCTGACA AG - #GCCTAATA    1510    - ATTCATGAAC AGGTCACAGT CAGAGGAGGG CTGGGCCCTG GGTGGCTTCA CA - #GATGTGGA    1570    - CTATTGGGAA CAGGGATCAC AGGGAGGKTG AGGTCAGSCG ACGGCGGCTG GG - #AGCAGTGC    1630    - AGCAGCAGGC AGGCGCTGCA GGGGAGTGAG GGTTCTGACA CTGGCCCACC CT - #GCAG  AG    1688    #        Glu    - TAC CCC TTC AAC CAG AGC CCC GTG GTG ACC CG - #C TCC TGC TCC AGC TCC    1736    Tyr Pro Phe Asn Gln Ser Pro Val Val Thr Ar - #g Ser Cys Ser Ser Ser    #     50    - TGT GTG GCC ACC GAC CCC GAC AGC ATC GGG GC - #C GCC CAC CTG ATC TTC    1784    Cys Val Ala Thr Asp Pro Asp Ser Ile Gly Al - #a Ala His Leu Ile Phe    # 70    - TGC TGC TTC CGA GAC CTC TGC AAC TCG GAA CT - #C TGAACCCAGG GCGGCAGGGC    1837    Cys Cys Phe Arg Asp Leu Cys Asn Ser Glu Le - #u    #                 80    - GGAAGGTGCT CCTCAGGCAC CTCCTCTCTG ACGGGGCCTG GCTCCACCTG TG - #ATCACCTC    1897    - CCCCTGCTTC CTGCTGCTGT GGCACAGCTC ACTCATGGGG TCTGAGGGGA GA - #GAAGCACA    1957    - CCAGGGGCGC CCTCTGCCTT CCATACCCCA CGCTTATAAA ACATAACTAA GC - #CAAGAGTG    2017    #   2031    - (2) INFORMATION FOR SEQ ID NO: 3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 540 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA to mRNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 22..330    -     (ix) FEATURE:              (A) NAME/KEY: mat.sub.-- - #peptide              (B) LOCATION: 88..330    -     (ix) FEATURE:              (A) NAME/KEY: TATA.sub.-- - #signal              (B) LOCATION: 505..511    #3:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #GTG CAG CTG CTG       51 ATG GCC TCT CGC TGG GCT    #Gln Leu Leula Ser Arg Trp Ala Val    15    - CTC GTG GCA GCC TGG AGC ATG GGC TGT GGT GA - #G GCC CTC AAG TGC TAC      99    Leu Val Ala Ala Trp Ser Met Gly Cys Gly Gl - #u Ala Leu Lys Cys Tyr    #         1    - ACC TGC AAG GAG CCC ATG ACC AGT GCT TCC TG - #C AGG ACC ATT ACC CGC     147    Thr Cys Lys Glu Pro Met Thr Ser Ala Ser Cy - #s Arg Thr Ile Thr Arg    #  20    - TGC AAG CCA GAG GAC ACA GCC TGC ATG ACC AC - #G CTG GTG ACG GTG GAG     195    Cys Lys Pro Glu Asp Thr Ala Cys Met Thr Th - #r Leu Val Thr Val Glu    #                 35    - GCA GAG TAC CCC TTC AAC CAG AGC CCC GTG GT - #G ACC CGC TCC TGC TCC     243    Ala Glu Tyr Pro Phe Asn Gln Ser Pro Val Va - #l Thr Arg Ser Cys Ser    #             50    - AGC TCC TGT GTG GCC ACC GAC CCC GAC AGC AT - #C GGG GCC GCC CAC CTG     291    Ser Ser Cys Val Ala Thr Asp Pro Asp Ser Il - #e Gly Ala Ala His Leu    #         65    - ATC TTC TGC TGC TTC CGA GAC CTC TGC AAC TC - #G GAA CTC TGAACCCAGG     340    Ile Phe Cys Cys Phe Arg Asp Leu Cys Asn Se - #r Glu Leu    #     80    - GCGGCAGGGC GGAAGGTGCT CCTCAGGCAC CTCCTCTCTG ACGGGGCCTG GC - #TCCACCTG     400    - TGATCACCTC CCCCTGCTTC CTGCTGCTGT GGCACAGCTC ACTCATGGGG TC - #TGAGGGGA     460    - GAGAAGCACA CCAGGGGCGC CCTCTGCCTT CCATACCCCA CGCTTATAAA AC - #ATAACTAA     520    #540               AAAA    - (2) INFORMATION FOR SEQ ID NO: 4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 103 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    #4:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    - Met Ala Ser Arg Trp Ala Val Gln Leu Leu Le - #u Val Ala Ala Trp Ser    10    - Met Gly Cys Gly Glu Ala Leu Lys Cys Tyr Th - #r Cys Lys Glu Pro Met    #   10    - Thr Ser Ala Ser Cys Arg Thr Ile Thr Arg Cy - #s Lys Pro Glu Asp Thr    #                 25    - Ala Cys Met Thr Thr Leu Val Thr Val Glu Al - #a Glu Tyr Pro Phe Asn    #             40    - Gln Ser Pro Val Val Thr Arg Ser Cys Ser Se - #r Ser Cys Val Ala Thr    #         55    - Asp Pro Asp Ser Ile Gly Ala Ala His Leu Il - #e Phe Cys Cys Phe Arg    #     70    - Asp Leu Cys Asn Ser Glu Leu    # 80    - (2) INFORMATION FOR SEQ ID NO: 5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #5:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 20               TTCT    - (2) INFORMATION FOR SEQ ID NO: 6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #6:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 20               TCCT    - (2) INFORMATION FOR SEQ ID NO: 7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #7:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #  18              CA    - (2) INFORMATION FOR SEQ ID NO: 8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #8:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 19               CAT    - (2) INFORMATION FOR SEQ ID NO: 9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #9:   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                23TAAT GGT    - (2) INFORMATION FOR SEQ ID NO: 10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #10:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #  18              GC    - (2) INFORMATION FOR SEQ ID NO: 11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #11:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 20               TTTG    - (2) INFORMATION FOR SEQ ID NO: 12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 17 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #12:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #   17             T    - (2) INFORMATION FOR SEQ ID NO: 13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #13:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #              26  TYAA YCARTC    - (2) INFORMATION FOR SEQ ID NO: 14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 26 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #14:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #              26  TYAA YCARAG    - (2) INFORMATION FOR SEQ ID NO: 15:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 29 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #15:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #            29    TGGA RTCNGGRTC    - (2) INFORMATION FOR SEQ ID NO: 16:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 29 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #16:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #            29    TGCT RTCNGGRTC    - (2) INFORMATION FOR SEQ ID NO: 17:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #17:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 19               GAG    - (2) INFORMATION FOR SEQ ID NO: 18:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #18:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 20               GCTG    - (2) INFORMATION FOR SEQ ID NO: 19:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 19 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #19:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    # 19               CGG    - (2) INFORMATION FOR SEQ ID NO: 20:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 21 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #20:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #21                TCAG T    - (2) INFORMATION FOR SEQ ID NO: 21:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 23 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #21:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                23CAAG GAG    - (2) INFORMATION FOR SEQ ID NO: 22:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 16 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #22:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #    16    - (2) INFORMATION FOR SEQ ID NO: 23:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 18 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #23:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #  18              TC    - (2) INFORMATION FOR SEQ ID NO: 24:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 37 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #24:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #      37          GTAC TTTTTTTTTT TTTTTTT    - (2) INFORMATION FOR SEQ ID NO: 25:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 48 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    -     (ix) FEATURE:              (A) NAME/KEY: modified.sub.-- - #base              (B) LOCATION: join(36..37, - # 41..42, 46..47)    #/mod.sub.-- base= iINFORMATION:    #25:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #                48ACGC GTCGACTAGT ACGGGNNGGG NNGGGNNG    - (2) INFORMATION FOR SEQ ID NO: 26:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 32 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: DNA    -    (iii) HYPOTHETICAL: NO    -    (iii) ANTI-SENSE: NO    #26:  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:    #          32      ACGC GTCGACTAGT AC    __________________________________________________________________________

I claim:
 1. An isolated and purified polypeptide comprising the peptidesequence of SEQ ID NO:1 or its salts and functional derivatives selectedfrom the group consisting of N-terminal or C-terminal acyl derivatives,or mixtures thereof.
 2. A process for the production of the polypeptideaccording to claim 1 comprising the following steps:a) adsorption ofurine, at pH acid, on kaolin and extraction with ammonia b) elution offraction (a) on Bio Rex 70 resin with ammonia c) elution of fraction (b)on DEAE Sepharose resin with acetate buffer d) elution of fraction (c)on CM Sepharose resin with acetate buffer e) elution of fraction (d) onHPLC C18 resin with a mixture of acetate buffer and acetonitrile f)elution of fraction (e) on DE-52 resin with acetate buffer g) elution offraction (f) on D-Zephyr resin with acetate buffer h) elution offraction (g) on HPLC C18 resin with a mixture of aqueous trifluoroaceticacid and acetonitrile i) elution of fraction (h) on D-Zephyr resin withacetate buffer.
 3. A process according to claim 2 wherein the urine ishuman urine.
 4. An isolate DNA molecule comprising the DNA sequencecoding for the polypeptide according to claim
 1. 5. An isolated DNAmolecule which hybridizes with the DNA molecule according to claim 4,wherein said hybridization is conducted with a horseradish peroxidaseoligoprobe and hybridized plaques formed are detected by enhancedchemiluminescence reagents under stringent hybridization conditionswherein said stringent hybridization conditions comprise 5×SSC, 0.02%SDS, 0.1% N-laurylsarcosine at 42° C.
 6. An isolated DNA moleculecomprising the nucleotide sequence of SEQ ID NO:
 2. 7. An isolated cDNAmolecule comprising the nucleotide sequence of SEQ ID NO:
 3. 8. Anexpression vector comprising the DNA molecule according to claim
 4. 9. Ahost cell transformed with an expression vector according to claim 8.10. An essentially pure protein, comprising the amino acid sequenceshown in SEQ ID NO:1, or its salts, obtainable through a processcomprising the following steps:a) adsorption of urine, at pH acid, onkaolin and extraction with ammonia b) elution of fraction (a) on ionexchange resin with ammonia c) elution of fraction (b) on ion exchangeresin with acetate buffer d) elution of fraction (c) on ion exchangeresin with acetate buffer e) elution of fraction (d) on ion exchangeresin with a mixture of acetate buffer and acetonitrile f) elution offraction (e) ion exchange resin with acetate buffer g) elution offraction (f) on ion exchange resin with acetate buffer h) elution offraction (g) on ion exchange resin with a mixture of aqueoustrifluoroacetic acid acetonitrile i) elution of fraction (h) on ionexchange resin with acetate buffer.
 11. A pharmaceutical compositioncomprising the protein according to claim 10, or its salts, or mixturesthereof in combination with one or more pharmaceutically acceptableeccipients or eluents.
 12. A composition comprising an effective amountof the polypeptide according to claim 1 to inhibit the binding ofTGF-alpha to its receptor and a pharmaceutically acceptable carrier. 13.An isolated and purified polypeptide according to claim 1 wherein saidpolypeptide is sulfated at position Tyr(39).
 14. The isolated DNAmolecule according to claim 5, wherein said hybridization conditionscomprise hybridization at 42° C. for 45 minutes.
 15. The isolated DNAmolecule according to claim 5, wherein said hybridization conditionscomprise hybridization at 50° C.